Flaviviruses are distributed worldwide and represent a global public health problem. Flaviviruses also have a significant impact as veterinary pathogens. Flavivirus pathogens include yellow fever (YF), dengue types 1-4 (DEN 1-4), Japanese encephalitis (JE), West Nile (WN), tick-borne encephalitis (TBE), and other viruses from the TBE serocomplex, such as Kyasanur Forest disease (KFD) and Omsk hemorrhagic fever (OHF) viruses. Vaccines against YF [live attenuated vaccine (LAV) strain 17D], JE [inactivated vaccines (INV) and LAV], and TBE (INV) are available. No licensed human vaccines are currently available against DEN and WN. Veterinary vaccines have been in use including, for example, vaccines against WN in horses (INV, recombinant and live chimeric vaccines), JE (INV and LAV) to prevent encephalitis in horses and stillbirth in pigs in Asia, louping ill flavivirus (INV) to prevent neurologic disease in sheep in the UK, and TBE (INV) used in farm animals in Czech Republic (INV) (Monath and Heinz, Flaviviruses, in Fields et al. Eds., Fields Virology, 3rd Edition, Philadelphia, New York, Lippincott-Raven Publishers, 1996, pp. 961-1034).
Tick-borne encephalitis (TBE) is the most important tick-borne viral disease of humans. It is endemic in parts of Europe and Northern Asia, causing more than 10,000 hospitalizations annually, with a case-fatality rate 0.5-1.5% in Europe and 6-40% in Siberia and the Far East. A significant proportion of patients suffer from long-lasting neuropsychiatric sequelae. Inactivated vaccines produced in chick embryo cell cultures have proven effective in preventing the disease. For example, an 86% vaccination coverage of the Austrian population (the highest among European countries) has resulted in an approximately 90% reduction of hospitalized cases (Heinz and Kunz, Arch. Virol. Suppl. 18:201-205, 2004). The inactivated vaccines are expensive and require three inoculations for primary immunization. Periodic boosters (every 2-5 years) are required to maintain immunity. Therefore, a less costly TBE vaccine, which is effective after one-two doses and provides durable, such as life-long immunity (similar to that achieved by YF 17D immunization) is needed, and indeed has been identified by the WHO as a major priority. Development of TBE LAV candidates in the past several decades by means of empirical or rational attenuation of TBE virus parent per se or chimerization of TBE or Langat (LGT, a naturally attenuated flavivirus that is closely related (serologically) to TBE) viruses with dengue 4 virus has faced difficulties due to problems with residual virulence of candidates and/or low immunogenicity/overattenuation (Wright et al., Vaccine 26:882-890, 2008; Maximova et al., J. Virol. 82:5255-5268, 2008; Rumyantsev et al., Vaccine 24:133-143, 2006; Kofler et al., Arch. Virol. Suppl. 18:191-200, 2004; and references therein).
Flaviviruses are small, enveloped, plus-strand RNA viruses transmitted primarily by arthropod vectors (mosquitoes or ticks) to natural hosts, which are primarily vertebrate animals, such as various mammals, including humans, and birds. The flavivirus genomic RNA molecule is about 11,000 nucleotides (nt) in length and encompasses a long open reading frame (ORF) flanked by 5′ and 3′ untranslated terminal regions (UTRs) of about 120 and 500 nucleotides in length, respectively. The ORF encodes a polyprotein precursor that is cleaved co- and post-translationally to generate individual viral proteins. The proteins are encoded in the order: C-prM/M-E-NS1-NS2A/2B-NS3-NS4A/4B-NS5, where C (core/capsid), prM/M (pre-membrane/membrane), and E (envelope) are the structural proteins, i.e., the components of viral particles, and the NS proteins are non-structural proteins, which are involved in intracellular virus replication. Flavivirus replication occurs in the cytoplasm. Upon infection of cells and translation of genomic RNA, processing of the polyprotein starts with translocation of the prM portion of the polyprotein into the lumen of endoplasmic reticulum (ER) of infected cells, followed by translocation of E and NS1 portions, as directed by the hydrophobic signals for the prM, E, and NS1 proteins. Amino-termini of prM, E, and NS1 proteins are generated by cleavage with cellular signalase, which is located on the luminal side of the ER membrane, and the resulting individual proteins remain carboxy-terminally anchored in the membrane. Most of the remaining cleavages, in the nonstructural region, are carried out by the viral NS2B/NS3 serine protease. The viral protease is also responsible for generating the C-terminus of the mature C protein found in progeny virions. Newly synthesized genomic RNA molecules and the C protein form a dense spherical nucleocapsid, which becomes surrounded by cellular membrane in which the E and prM proteins are embedded. The mature M protein is produced by cleavage of prM shortly prior to virus release by cellular furin or a similar protease. E, the major protein of the envelope, is the principal target for neutralizing antibodies, the main correlate of immunity against flavivirus infection. Virus-specific cytotoxic T-lymphocyte (CTL) response is the other key attribute of immunity. Multiple CD8+ and CD4+ CTL epitopes have been characterized in various flavivirus structural and non-structural proteins. In addition, innate immune responses contribute to both virus clearance and regulating the development of adaptive immune responses and immunologic memory.
In addition to the inactivated (INV) and live-attenuated (LAV) vaccines against flaviviruses discussed above, other vaccine platforms have been developed. One example is based on chimeric flaviviruses that include yellow fever virus capsid and non-structural sequences and prM-E proteins from other flaviviruses, to which immunity is sought. This technology has been used to develop vaccine candidates against dengue (DEN), Japanese encephalitis (JE), West Nile (WN), and St. Louis encephalitis (SLE) viruses (see, e.g., U.S. Pat. Nos. 6,962,708 and 6,696,281). Yellow fever virus-based chimeric flaviviruses have yielded highly promising results in clinical trials.
Another flavivirus vaccine platform is based on the use of pseudoinfectious virus (PIV) technology (Mason et al., Virology 351:432-443, 2006; Shustov et al., J. Virol. 21:11737-11748, 2007; Widman et al., Adv. Virus. Res. 72:77-126, 2008; Suzuki et al., J. Virol. 82:6942-6951, 2008; Suzuki et al., J. Virol. 83:1870-1880, 2009; Ishikawa et al., Vaccine 26:2772-2781, 2008; Widman et al., Vaccine 26:2762-2771, 2008). PIVs are replication-defective viruses attenuated by a deletion(s). Unlike live flavivirus vaccines, they undergo a single round replication in vivo (or optionally limited rounds, for two-component constructs; see below), which may provide benefits with respect to safety. PIVs also do not induce viremia and systemic infection. Further, unlike inactivated vaccines, PIVs mimic whole virus infection, which can result in increased efficacy due to the induction of robust B- and T-cell responses, higher durability of immunity, and decreased dose requirements. Similar to whole viruses, PIV vaccines target antigen-presenting cells, such as dendritic cells, stimulate toll-like receptors (TLRs), and induce balanced Th1/Th2 immunity. In addition, PIV constructs have been shown to grow to high titers in substrate cells, with little or no cytopathic effect (CPE), allowing for high-yield manufacture, optionally employing multiple harvests and/or expansion of infected substrate cells.
The principles of the PIV technology are illustrated in FIGS. 1 and 2. There are two variations of the technology. In the first variation, a single-component pseudoinfectious virus (s-PIV) is constructed with a large deletion in the capsid protein (C), rendering mutant virus unable to form infectious viral particles in normal cells (FIG. 1). The deletion does not remove the first ˜20 codons of the C protein, which contain an RNA cyclization sequence, and a similar number of codons at the end of C, which encode a viral protease cleavage site and the signal peptide for prM. The s-PIV can be propagated, e.g., during manufacture, in substrate (helper) cell cultures in which the C protein is supplied in trans, e.g., in stably transfected cells producing the C protein (or a larger helper cassette including C protein), or in cells containing an alphavirus replicon [e.g., a Venezuelan equine encephalitis virus (VEE) replicon] expressing the C protein or another intracellular expression vector expressing the C protein. Following inoculation in vivo, e.g., after immunization, the PIV undergoes a single round of replication in infected cells in the absence of trans-complementation of the deletion, without spread to surrounding cells. The infected cells produce empty virus-like particles (VLPs), which are the product of the prM-E genes in the PIV, resulting in the induction of neutralizing antibody response. A T-cell response should also be induced via MHCI presentation of viral epitopes. This approach has been applied to YF 17D virus and WN viruses and WN/JE and WN/DEN2 chimeric viruses (Mason et al., Virology 351:432-443, 2006;
Suzuki et al., J. Virol. 83:1870-1880, 2009; Ishikawa et al., Vaccine 26:2772-2781, 2008; Widman et al., Vaccine 26:2762-2771, 2008; WO 2007/098267; WO 2008/137163).
In the second variation, a two-component PIV (d-PIV) is constructed (FIG. 2). Substrate cells are transfected with two defective viral RNAs, one with a deletion in the C gene and another lacking the prM-E envelope protein genes. The two defective genomes complement each other, resulting in accumulation of two types of PIVs in the cell culture medium (Shustov et al., J. Virol. 21:11737-11748, 2007; Suzuki et al., J. Virol. 82:6942-6951, 2008). Optionally, the two PIVs can be manufactured separately in appropriate helper cell lines and then mixed in a two-component formulation. The latter may offer an advantage of adjusting relative concentrations of the two components, increasing immunogenicity and efficacy. This type of PIV vaccine should be able to undergo a limited spread in vivo due to coinfection of some cells at the site of inoculation with both components. The spread is expected to be self-limiting as there are more cells in tissues than viral particles produced by initially coinfected cells. In addition, a relatively high MOI is necessary for efficient co-infection, and cells outside of the inoculation site are not expected to be efficiently coinfected (e.g., in draining lymph nodes). Cells infected with the AC PIV alone produce the highly immunogenic VLPs. Coinfected cells produce the two types of packaged defective viral particles, which also stimulate neutralizing antibodies. The limited infection is expected to result in a stronger neutralizing antibody response and T-cell response compared to s-PIVs. To decrease chances of recombination during manufacture or in vivo, including with circulating flaviviruses, viral sequences can be modified in both s-PIVs and d-PIVs using, e.g., synonymous codon replacements, to reduce nucleotide sequence homologies, and mutating the complementary cyclization 5′ and 3′ elements.